The Mos protein kinase, originally identified as the gene product of the v- mos oncogen of moloney Sarcoma Virus, plays three key roles in cell cycle regulation: first, as an essential protein for resting oocytes to progress to germinal vesicle breakdown (GVBD); second, as an essential protein for progression from meiosis I to meiosis II; and third, as an essential component of cytostatic factor (CSF). The overall objective of this grant is to elucidate further the interactions between Mos, cdc2 and cyclin B1 in cell cycle control. The first specific aim examines newly-identified Mos-and cdc2-interacting proteins. Using the yeast two-hybrid system, we have identified novel proteins designated PP5, Vip-alpha and Vip-beta, and Cjm1 for which full- length cDNAs have been recovered and sequenced. In addition, we have also identified GST pi, a glutathione-S-transferase, as a potentially important regulator of meiotic maturation. These novel proteins will be characterized with respect to: interactions with Mos or cdc2; effects on MPF and MEK activation during meiotic maturation; and as potential cell cycle regulatory proteins in mammalian cells. The second specific aim examines the integration of Mos in signal transduction pathways. Mos has been implicated as a MEK kinase, and we will examine the role of Mos or Mos-associated proteins ina the activation of MEK. We will also examine whether mos may be regulated by targeting it to different intracellular compartments, which may alter its accessibility to key effectors. Interactions between Mos and a member of the PKC family, zeta[unreadable]KC, which plays an important role in the control of meiotic maturation in oocytes, will also be investigate. The role of Mos ina the progression from meiosis I to meiosis II will be further examined with regard to regulatory phosphorylation sites of cdc2. In the third specific aim, the role of phosphorylation in regulating the subcellular localization of cyclin B1 will be examined. We will continue exciting preliminary experiments demonstrating that phosphorylation may be required to direct cyclin B1 to a perinuclear localization prior to breakdown of the nuclear membrane. These experiments will also examine the possibility that phosphorylation may regulate athe function of a "cytoplasmic retention signal" which spans four of the identified phosphorylaiton sites. The demonstration that phosphorylation regulates the subcellular localization of cyclin B1 would be of fundamental importance to our understanding of cell cycle control.